Gap junctions allow intercellular communication between adjacent cells and are formed from hexameric structures called connexons. Each connexon comprises a hemichannel and docks with a connexon on an apposing cell across a narrow gap to form a junction. Ions and metabolites are exchanged between cells across these junctions. Of the 21 human connexin (Cx) genes discovered, four are considered principal isoforms in cardiac tissue: Cx37, Cx40, Cx43 and Cx45. Gap junctions in cardiac tissue act as the electrical conduits of the heart, helping to maintain a uniform action potential for a proper heartbeat. Disruption of these junctions can lead to fatal arrhythmias, especially in post-myocardial infarction patients. The molecular basis for channel closure and regulation is not well understood. These gap junctions are also potential antiarrhythmic drug targets, but pharmaceutical developments are hindered due to the lack of high resolution three dimensional models. I am proposing an integrated approach to study cardiac connexin structures which employs recombinant protein expression, electron cryo-microscopy (cryoEM), EPR spectroscopy, and molecular dynamics (MD) simulations to explore the structure and conformational dynamics of cardiac gap junction membrane channels, focusing on the most abundant heart isoforms, Cx40 and Cx43. Cx40 is enriched in the His-Purkinje fibers whereas Cx43 is found throughout the myocardium. Although they possess a high degree of sequence similarity, Cx40 and Cx43 have markedly different expression and physiological profiles. High resolution structures would assist in explaining the immense functional data that has accumulated over the last four decades and can pave the way for rationalized drug design. The goal of this proposal is to comparatively characterize Cx40 and Cx43 at the atomic level using cryoEM, EPR, and MD in order to establish the molecular determinants of their functional differences and to derive a detailed molecular description of channel closure and regulation.